Nanovesicle derived from Proteus genus bacteria, and use thereof

ABSTRACT

Provided are vesicles derived from bacteria belonging to the genus Proteus and a use thereof. The inventors of the present invention experimentally confirmed that the vesicles was significantly reduced in samples of patients with cancers, allergic-respiratory diseases, cardiovascular diseases, metabolic diseases, or neuropsychiatric diseases, as compared to that of normal people, and the vesicles inhibited the secretion of inflammatory mediators due to pathogenic vesicles and also exhibited anticancer efficacy. Therefore, it is anticipated that the vesicles derived from bacteria belonging to the genus Proteus, according to the present invention, may be usefully used for the development of a method of diagnosing cancer, cardiovascular diseases, metabolic diseases, neuropsychiatric diseases, allergic-respiratory diseases, and inflammatory bowel diseases, and a composition for prevention, treatment, and/or alleviation.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.16/362,176, filed on Mar. 22, 2019, which is a continuation ofPCT/KR2018/007144, filed Jun. 25, 2018, which claims the benefit ofpriority from Korean Patent Application No. 10-2017-0083047, filed Jun.30, 2017 and Korean Patent Application No. 10-2018-0072307, filed Jun.22, 2018, the contents of each of which are incorporated herein byreference in its entirety.

SEQUENCE LISTING

The Sequence Listing submitted in text format (.txt) filed on Feb. 15,2021, named “SequenceListing.txt”, created on Feb. 12, 2021 (719 bytes),is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to nanovesicles derived from bacteriabelonging to the genus Proteus and a use thereof, and more particularly,to a method of diagnosing cancers, cardiovascular diseases, metabolicdiseases, neuropsychiatric diseases, allergic-respiratory diseases, andinflammatory bowel diseases, which are characterized by chronicinflammation, by using nanovesicles derived from bacteria belonging tothe genus Proteus, and a preventive or therapeutic composition includingthe nanovesicles.

BACKGROUND ART

In the 21^(st) century, the importance of acute infectious diseases,which have been recognized as epidemics in the past, has become lessimportant. On the other hand, a disease pattern has been changed suchthat chronic diseases accompanied by immune dysfunction occurring due toincompatibility between humans and microbiomes are considered as a majordisease that determines the quality of life and human lifespan. In the21^(st) century, intractable chronic diseases are characterized bychronic inflammatory responses caused by immune dysfunction due torepetitive exposure to causative factors according to lifestyle changes,and as examples thereof, cancer, cardiovascular diseases, metabolicdiseases, neuropsychiatric diseases, allergic-respiratory diseases,inflammatory bowel diseases, and the like are becoming serious problemsin public health.

As immune responses to bacteria-derived causative factors, a T helper 17(Th17) immune response characterized by the secretion of interleukin-17cytokines is important, and an innate immune response such as thesecretion of interleukin-6 (IL-6) due to bacterial factors plays acrucial role in differentiation into Th17 cells. In addition, it isknown that inflammation occurs upon exposure to bacterial causativefactors, and in this process, inflammatory mediators such as tumornecrosis factor-α (TNF-α) are secreted, thus causing the onset ofcancer, cardiovascular diseases, metabolic diseases, neuropsychiatricdiseases, allergic-respiratory diseases, inflammatory bowel diseases,and the like.

It is known that the number of microorganisms symbiotically living inthe human body is 100 trillion which is 10 times that of human cells,and the number of genes of microorganisms exceeds 100 times that ofhumans. Bacteria and archaea symbiotically living in the human bodysecrete nanometer-sized vesicles to exchange information about genes,proteins, and the like with other cells. The mucous membranes form aphysical barrier membrane that does not allow particles with a size of200 nm or more to pass therethrough, and thus bacteria symbioticallyliving in the mucous membranes are unable to pass therethrough, butbacteria-derived vesicles have a size of approximately 100 nm or lessand thus relatively freely pass through the mucous membranes and areabsorbed into epithelial cells, thus inducing inflammatory responses,and are also absorbed into the human body through lymphatic vessels.Bacteria-derived vesicles that are locally secreted from bacteriasymbiotically living in the human body are absorbed into epithelialcells of the mucous membrane to thereby induce a local inflammatoryresponse, and pathogenic bacteria-derived vesicles absorbed into thehuman body are distributed to respective organs through blood to therebyincrease inflammatory responses in the organs, thus aggravatingdiseases.

Bacteria belonging to the genus Proteus, which are anaerobicGram-negative bacilli, convert urea into ammonia through a urease tothereby alkalize urine. Among the above bacteria, Proteus mirabilis,which is known as a pathogenic bacterium, accounts for 90% of the casesof infection with bacteria belonging to the genus Proteus in humans. Thebacteria belonging to the genus Proteus are known to symbiotically livein the digestive organs of humans and also widely inhabit an environmentsuch as soil, water, and the like.

Meanwhile, inventions that relate to treatment of diseases usingextracellular vesicles derived from bacteria belonging to the genusBacillus or lactic acid bacteria are disclosed (KR 10-1862507 and KR10-1726488), but to date, the fact that bacteria belonging to the genusProteus extracellularly secrete vesicles has not been reported, andparticularly there are no reports of the use of bacteria belonging tothe genus Proteus in the diagnosis and treatment of diseases such ascancer, cardiovascular diseases, metabolic diseases, neuropsychiatricdiseases, allergic-respiratory diseases, inflammatory bowel diseases,and the like.

Therefore, it was confirmed in the present invention that the amount ofvesicles derived from bacteria belonging to the genus Proteus wassignificantly reduced in clinical samples of patients with cancer,cardiovascular diseases, metabolic diseases, neuropsychiatric diseases,allergic-respiratory diseases, and inflammatory bowel diseases, ascompared to that of normal people, and thus the diseases could bediagnosed. In addition, it was confirmed that vesicles isolated fromculture broths of bacteria belonging to the genus Proteus could be usedas a composition for preventing or treating cancer, cardiovasculardiseases, metabolic diseases, neuropsychiatric diseases,allergic-respiratory diseases, and inflammatory bowel diseases.

DESCRIPTION OF EMBODIMENTS Technical Problem

As a result of having conducted intensive studies to address theabove-described conventional problems, the inventors of the presentinvention confirmed through metagenomic analysis that the amount ofvesicles derived from bacteria belonging to the genus Proteus wassignificantly reduced, as compared to that of normal people, in samplesderived from patients with cancers such as stomach cancer, colorectalcancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer,breast cancer, ovarian cancer, bladder cancer, prostate cancer,lymphoma, a brain tumor, and the like; cardiovascular diseases such asmyocardial infarction, cardiomyopathy, atrial fibrillation, variantangina, a stroke, and the like; metabolic diseases such as diabetes, andthe like; neuropsychiatric diseases such as Parkinson's disease,depression, and the like; allergic-respiratory diseases such as atopicdermatitis, asthma, chronic obstructive pulmonary disease (COPD), andthe like; or inflammatory bowel diseases such as irritable bowelsyndrome, inflammatory enteritis, and the like.

It was also confirmed that, when vesicles were isolated from Proteusmirabilis, which is a bacterium belonging to the genus Proteus, andinflammatory cells were treated with the vesicles, the secretion ofTNF-α due to pathogenic vesicles was significantly inhibited, and as aresult of evaluating anticancer efficacy, when mouse cancer diseasemodels were orally administered vesicles derived from Proteus mirabilis,the onset of cancer was significantly inhibited, and thus the presentinvention was completed based on these findings.

Therefore, an object of the present invention is to provide a method ofproviding information for the diagnosis of one or more diseases selectedfrom the group consisting of stomach cancer, colorectal cancer, livercancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer, lymphoma, a braintumor, chronic obstructive pulmonary disease, atopic dermatitis,irritable bowel syndrome, inflammatory enteritis, asthma, myocardialinfarction, cardiomyopathy, variant angina, atrial fibrillation, astroke, diabetes, Parkinson's disease, and depression.

Another object of the present invention is to provide a composition forpreventing, treating, and/or alleviating one or more diseases selectedfrom the group consisting of stomach cancer, colorectal cancer, livercancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer, lymphoma, a braintumor, chronic obstructive pulmonary disease, atopic dermatitis,irritable bowel syndrome, inflammatory enteritis, asthma, myocardialinfarction, cardiomyopathy, variant angina, atrial fibrillation, astroke, diabetes, Parkinson's disease, and depression, the compositionincluding vesicles derived from bacteria belonging to the genus Proteusas an active ingredient.

However, technical problems to be achieved by the present invention arenot limited to the above-described technical problems, and otherunmentioned technical problems will become apparent from the followingdescription to those of ordinary skill in the art.

Technical Solution

To achieve the above-described objects of the present invention, thepresent invention provides a method of providing information for thediagnosis of one or more diseases selected from the group consisting ofcancer, a cardiovascular disease, a metabolic disease, aneuropsychiatric disease, an allergic-respiratory disease, and aninflammatory bowel disease, the method including the followingprocesses:

(a) extracting DNA from vesicles isolated from normal people-derivedsamples and subject-derived samples;

(b) performing polymerase chain reaction (PCR) on the extracted DNA byusing a pair of primers prepared based on a gene sequence present in 16SrDNA to obtain respective PCR products; and

(c) determining a case, in which the amount of vesicles derived frombacteria belonging to the genus Proteus is lower than that of normalpeople, as one or more diseases selected from the group consisting ofcancer, a cardiovascular disease, a metabolic disease, aneuropsychiatric disease, an allergic-respiratory disease, and aninflammatory bowel disease, through quantitative analysis of the PCRproducts.

The present invention also provides a method of diagnosing one or morediseases selected from the group consisting of cancer, a cardiovasculardisease, a metabolic disease, a neuropsychiatric disease, anallergic-respiratory disease, and an inflammatory bowel disease, themethod including the following processes:

(a) extracting DNA from vesicles isolated from normal people-derivedsamples and subject-derived samples;

(b) performing PCR on the extracted DNA by using a pair of primersprepared based on a gene sequence present in 16S rDNA to obtainrespective PCR products; and

(c) determining a case, in which the amount of vesicles derived frombacteria belonging to the genus Proteus is lower than that of normalpeople, as one or more diseases selected from the group consisting ofcancer, a cardiovascular disease, a metabolic disease, aneuropsychiatric disease, an allergic-respiratory disease, and aninflammatory bowel disease, through quantitative analysis of the PCRproducts.

In another embodiment of the present invention, in process (a), thesamples may be blood, urine, or stool.

The present invention also provides a pharmaceutical composition forpreventing or treating one or more diseases selected from the groupconsisting of cancer, a cardiovascular disease, a metabolic disease, aneuropsychiatric disease, an allergic-respiratory disease, and aninflammatory bowel disease, the pharmaceutical composition includingvesicles derived from bacteria belonging to the genus Proteus as anactive ingredient.

The present invention also provides an inhalant composition forpreventing or treating one or more diseases selected from the groupconsisting of cancer, a cardiovascular disease, a metabolic disease, aneuropsychiatric disease, an allergic-respiratory disease, and aninflammatory bowel disease, the inhalant composition including vesiclesderived from bacteria belonging to the genus Proteus as an activeingredient.

The present invention also provides a composition for preventing oralleviating one or more diseases selected from the group consisting ofcancer, a cardiovascular disease, a metabolic disease, aneuropsychiatric disease, an allergic-respiratory disease, and aninflammatory bowel disease, the composition including vesicles derivedfrom bacteria belonging to the genus Proteus as an active ingredient.

In one embodiment of the present invention, the composition forpreventing or alleviating the above-described diseases may be a foodcomposition or a cosmetic composition.

In another embodiment of the present invention, the food composition maybe a health functional food composition.

The present invention also provides a method of preventing or treatingone or more diseases selected from the group consisting of cancer, acardiovascular disease, a metabolic disease, a neuropsychiatric disease,an allergic-respiratory disease, and an inflammatory bowel disease, themethod including administering, to an individual, a pharmaceuticalcomposition including vesicles derived from bacteria belonging to thegenus Proteus as an active ingredient.

The present invention also provides a use of vesicles derived frombacteria belonging to the genus Proteus for preventing or treating oneor more diseases selected from the group consisting of cancer, acardiovascular disease, a metabolic disease, a neuropsychiatric disease,an allergic-respiratory disease, and an inflammatory bowel disease.

In one embodiment of the present invention, the cancer may be stomachcancer, colorectal cancer, liver cancer, bile duct cancer, pancreaticcancer, lung cancer, breast cancer, ovarian cancer, bladder cancer,prostate cancer, lymphoma, or a brain tumor.

In another embodiment of the present invention, the cardiovasculardisease may be myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, or a stroke.

In another embodiment of the present invention, the metabolic diseasemay be diabetes.

In another embodiment of the present invention, the neuropsychiatricdisease may be Parkinson's disease or depression.

In another embodiment of the present invention, the allergic-respiratorydisease may be atopic dermatitis, asthma, or chronic obstructivepulmonary disease.

In another embodiment of the present invention, the inflammatory boweldisease may be irritable bowel syndrome or inflammatory enteritis.

In another embodiment of the present invention, the vesicles may have anaverage diameter of 10 nm to 200 nm.

In another embodiment of the present invention, the vesicles may benaturally or artificially secreted from bacteria belonging to the genusProteus.

In another embodiment of the present invention, the vesicles derivedfrom bacteria belonging to the genus Proteus may be secreted fromProteus mirabilis.

Advantageous Effects of Invention

The inventors of the present invention confirmed that althoughintestinal bacteria would not be absorbed into the body,bacteria-derived vesicles were absorbed into the body through epithelialcells and distributed systemically, and excreted outside the bodythrough the kidneys, the liver, and the lungs, and confirmed throughmetagenomic analysis of bacteria-derived vesicles present in blood,urine, stool, or the like of patients that vesicles derived frombacteria belonging to the genus Proteus, which are present in blood,urine, or stool of patients with cancers such as stomach cancer,colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, a brain tumor, and the like; cardiovascular diseasessuch as myocardial infarction, cardiomyopathy, atrial fibrillation,variant angina, a stroke, and the like; metabolic diseases such asdiabetes, and the like; neuropsychiatric diseases such as Parkinson'sdisease, depression, and the like; allergic-respiratory diseases such asatopic dermatitis, asthma, chronic obstructive pulmonary disease (COPD),and the like; inflammatory bowel diseases such as irritable bowelsyndrome, inflammatory enteritis, and the like, was significantlyreduced as compared to that of normal people. In addition, it wasobserved that, when Proteus mirabilis, which is one species of bacteriabelonging to the genus Proteus, was cultured in vitro, and vesicles wereisolated therefrom and administered to in vitro inflammatory cells, thesecretion of inflammatory mediators due to pathogenic vesicles wassignificantly inhibited, and when the vesicles were orally administeredto mice, the onset of cancer was significantly inhibited, and thus it isanticipated that the vesicles derived from bacteria belonging to thegenus Proteus according to the present invention can be usefully used ina method of diagnosing cancers such as stomach cancer, colorectalcancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer,breast cancer, ovarian cancer, bladder cancer, prostate cancer,lymphoma, a brain tumor, and the like; cardiovascular diseases such asmyocardial infarction, cardiomyopathy, atrial fibrillation, variantangina, a stroke, and the like; metabolic diseases such as diabetes, andthe like; neuropsychiatric diseases such as Parkinson's disease,depression, and the like; allergic-respiratory diseases such as atopicdermatitis, asthma, chronic obstructive pulmonary disease, and the like;inflammatory bowel diseases such as irritable bowel syndrome,inflammatory enteritis, and the like, and a composition for prevention,treatment, and/or alleviation, such as a food, an inhalant, a cosmetic,a drug, or the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A illustrates images showing distribution patterns of bacteria andvesicles according to time after bacteria and bacteria-derived vesicles(EV) were orally administered to mice.

FIG. 1B illustrates images obtained as a result of evaluating in vivodistribution patterns of bacteria and vesicles in blood, the kidneys,the liver, and various organs which were extracted at 12 hours afterbacteria and bacteria-derived vesicles (EV) were orally administered tomice.

FIG. 2 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin stool, blood, and urine samples of patients with stomach cancer andnormal people.

FIG. 3 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin stool and urine samples of patients with colorectal cancer and normalpeople.

FIG. 4 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood samples of patients with liver cancer, bile duct cancer, orpancreatic cancer, and normal people.

FIG. 5 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood samples of patients with lung cancer and normal people.

FIG. 6 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin urine samples of patients with breast cancer or ovarian cancer, andnormal people.

FIG. 7 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood and urine samples of patients with bladder cancer and normalpeople.

FIG. 8 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin urine samples of patients with prostate cancer and normal people.

FIG. 9 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood samples of patients with lymphoma or a brain tumor and normalpeople.

FIG. 10 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood samples of patients with diabetes and normal people.

FIG. 11 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood samples of patients with myocardial infarction, cardiomyopathy,atrial fibrillation, or variant angina and normal people.

FIG. 12 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood samples of patients with a stroke and normal people.

FIG. 13 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin urine samples of patients with Parkinson's disease or depression andnormal people.

FIG. 14 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood samples of patients with atopic dermatitis and normal people.

FIG. 15 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin blood samples of patients with asthma or chronic obstructivepulmonary disease (COPD) and normal people.

FIG. 16 illustrates results of comparing distribution patterns ofvesicles derived from bacteria belonging to the genus Proteus aftermetagenomic analysis was performed on bacteria-derived vesicles presentin stool samples of patients with irritable bowel syndrome, inflammatoryenteritis, and normal people.

FIG. 17A illustrates a microscope image showing observation results ofvesicles isolated from a culture broth obtained after Proteus mirabiliswas cultured in vitro.

FIG. 17B illustrates results of measuring the size of vesicles isolatedfrom a Proteus mirabilis culture broth, by dynamic light scattering.

FIGS. 18A and 18B illustrate results of evaluating an effect of E. colivesicles (E. coli EV) on the secretion of IL-6 (FIG. 18A) and TNF-α(FIG. 18B), which are inflammatory mediators, in the case ofpretreatment with vesicles derived from bacteria belonging to the genusProteus prior to treatment with the E. coli vesicles, which arepathogenic vesicles, to evaluate an anti-inflammatory effect of Proteusmirabilis-derived vesicles.

FIG. 19 illustrate results of evaluating an effect of E. coli vesicles(E. coli EV) on the secretion of TNF-α, in the case of pretreatment withProteus mirabilis (PMR)-derived vesicles isolated from people prior totreatment with E. coli vesicles, which are pathogenic vesicles, tocompare effects of vesicles derived from various strains with theanti-inflammatory effect of Proteus mirabilis-derived vesicles (NC:negative control; PC: positive control; L. plantarum: Lactobacillusplantarum).

FIG. 20 illustrate results of evaluating an effect of E. coli vesicleson the secretion of TNF-α, in the case of pretreatment with heat-treatedor acid-treated Proteus mirabilis (PMR)-derived vesicles beforetreatment with the E. coli vesicles (E. coli EV), which are pathogenicvesicles, to evaluate an effect of heat treatment or acid treatment onthe anti-inflammatory effect of Proteus mirabilis-derived vesicles (NC:negative control; PC: positive control; L. plantarum: Lactobacillusplantarum).

FIG. 21A illustrates a protocol by which Proteus mirabilis-derivedvesicles were administered to mice to evaluate the anticancer efficacyof the Proteus mirabilis-derived vesicles.

FIG. 21B illustrates results of evaluating an effect of Proteusmirabilis-derived vesicles on tumorigenesis due to cancer cells whenorally administered to mice.

BEST MODE

The present invention relates to vesicles derived from bacteriabelonging to the genus Proteus and a use thereof.

The inventors of the present invention confirmed through metagenomicanalysis that the amount of vesicles derived from bacteria belonging tothe genus Proteus was significantly reduced, as compared to that ofnormal people, in samples derived from patients with cancers such asstomach cancer, colorectal cancer, liver cancer, bile duct cancer,pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladdercancer, prostate cancer, lymphoma, a brain tumor, and the like;cardiovascular diseases such as myocardial infarction, cardiomyopathy,variant angina, atrial fibrillation, a stroke, and the like; metabolicdiseases such as diabetes, and the like; neuropsychiatric diseases suchas Parkinson's disease, depression, and the like; allergic-respiratorydiseases such as atopic dermatitis, asthma, chronic obstructivepulmonary disease, and the like; or inflammatory bowel diseases such asirritable bowel syndrome, inflammatory enteritis, and the like, and thuscompleted the present invention based on these findings.

Therefore, the present invention provides a method of providinginformation for the diagnosis of one or more diseases selected from thegroup consisting of stomach cancer, colorectal cancer, liver cancer,bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovariancancer, bladder cancer, prostate cancer, lymphoma, a brain tumor,chronic obstructive pulmonary disease, atopic dermatitis, irritablebowel syndrome, inflammatory enteritis, asthma, myocardial infarction,cardiomyopathy, variant angina, atrial fibrillation, a stroke, diabetes,Parkinson's disease, and depression, the method including the followingprocesses:

(a) extracting DNA from vesicles isolated from normal people-derivedsamples and subject-derived samples;

(b) performing PCR on the extracted DNA by using a pair of primersprepared based on a gene sequence present in 16S rDNA to obtainrespective PCR products; and

(c) determining a case, in which the amount of vesicles derived frombacteria belonging to the genus Proteus is lower than that of normalpeople, as one or more diseases selected from the group consisting ofstomach cancer, colorectal cancer, liver cancer, bile duct cancer,pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladdercancer, prostate cancer, lymphoma, a brain tumor, chronic obstructivepulmonary disease, atopic dermatitis, irritable bowel syndrome,inflammatory enteritis, asthma, myocardial infarction, cardiomyopathy,variant angina, atrial fibrillation, a stroke, diabetes, Parkinson'sdisease, and depression, through quantitative analysis of the PCRproducts.

The term “diagnosis” as used herein means, in a broad sense, determiningconditions of disease of a patient in all aspects. Content of thedetermination includes disease name, the cause of a disease, the type ofdisease, the severity of disease, detailed aspects of syndrome, thepresence or absence of complications, prognosis, and the like. In thepresent invention, diagnosis means determining the presence or absenceof the onset of cancer, a cardiovascular disease, a metabolic disease, aneuropsychiatric disease, an allergic-respiratory disease, or aninflammatory bowel disease, the severity of disease, and the like.

The term “cancer,” which is a diagnostic target disease of the presentinvention, means malignant tumors that grow rapidly while infiltratinginto the surrounding tissues and diffuse or transit to each part of thebody, and thus are life-threatening. Cells, which are the smallest unitof the body, normally divide and grow by the regulatory function ofcells themselves, and when the lifespan of cells end or cells getdamaged, they themselves die and thus maintain a balance in an overallnumber of cells. However, when cells have a problem with such aregulatory function thereof, due to various causes, abnormal cells,which would normally die, excessively proliferate, and infiltrate intothe surrounding tissues and organs to thereby form a mass, resulting indestruction or modification of existing structures. In the presentinvention, the cancer may be preferably stomach cancer, colorectalcancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer,breast cancer, ovarian cancer, bladder cancer, prostate cancer,lymphoma, a brain tumor, but the present invention is not limitedthereto.

The term “cardiovascular disease” as used herein means the onset of adisease in the cardiovascular system of a mammal, and examples thereofinclude heart diseases such as myocardial infarction, cardiomyopathy,angina, arrhythmia, and the like; vasculitis; cerebrovascular diseasessuch as dementia, a stroke, and the like; and the like. In the presentinvention, the cardiovascular disease preferably includes, but is notlimited to, myocardial infarction, cardiomyopathy, variant angina,atrial fibrillation, or a stroke.

The term “metabolic disease” as used herein refers to a disease in whichcomplications occur in various organs due to metabolic disorders in thebody of a mammal, and examples thereof include metabolic disorders suchas hyperlipidemia, diabetes, and the like, complications thereof, andthe like. In the present invention, the metabolic disease preferablyincludes, but is not limited to, diabetes.

The term “neuropsychiatric disease” as used herein refers to a diseaseoccurring in the nervous system and brain of a mammal, and examplesthereof include brain diseases such as Parkinson's disease, dementia,and the like; mental diseases such as depression, obsessive-compulsivedisorder, schizophrenia, and the like; and the like. In the presentinvention, the neuropsychiatric disease preferably includes, but is notlimited to, Parkinson's disease and depression.

The term “allergic disease” as used herein refers to a disease caused byallergic mechanisms in mammals, and examples thereof include skindiseases such as atopic dermatitis, respiratory diseases such asallergic rhinitis and asthma, and the like. In the present invention,the allergic disease preferably includes, but is not limited to, atopicdermatitis and asthma.

The term “respiratory disease” as used herein refers to a diseaseoccurring in the respiratory system of a mammal, and examples thereofinclude rhinitis, asthma, chronic obstructive pulmonary disease, and thelike. In the present invention, the respiratory disease preferablyincludes, but is not limited to, asthma and chronic obstructivepulmonary disease.

The term “inflammatory bowel disease” as used herein refers tounidentified chronic inflammation occurring in the intestines, and in abroad sense, may include all inflammatory diseases occurring in theintestines, such as infectious enteritis and ischemic bowel disease suchas bacterial, viral, amoebic, or tuberculous enteritis, and the like;radiation enteritis; and the like. In the present invention, theinflammatory bowel disease preferably includes, but is not limited to,irritable bowel syndrome and inflammatory enteritis.

The term “nanovesicles” or “vesicles” as used herein refers tostructures consisting of nano-sized membranes secreted by variousbacteria. Gram-negative bacteria-derived vesicles, or outer membranevesicles (OMVs) contain lipopolysaccharides, toxic proteins, bacterialDNA and RNA, and various metabolites, and gram-positive bacteria-derivedvesicles also contain peptidoglycan and lipoteichoic acid, which arecell wall components of bacteria, in addition to proteins and nucleicacids. In the present invention, nanovesicles or vesicles are naturallysecreted or artificially produced in bacteria belonging to the genusProteus, and have an average diameter of 10 nm to 200 nm.

The vesicles may be isolated from a culture broth containing bacteriabelonging to the genus Proteus by using one or more methods selectedfrom centrifugation, ultracentrifugation, extrusion, ultrasonicdegradation, cell lysis, homogenization, freezing-thawing,electroporation, mechanical degradation, chemical treatment, filtrationusing a filter, gel filtration chromatography, free-flowelectrophoresis, and capillary electrophoresis. In addition, theisolation methods may further include washing for the removal ofimpurities, concentration of obtained vesicles, and the like.

The term “metagenome” as used herein refers to the total of genomesincluding all viruses, bacteria, fungi, and the like in isolated regionssuch as soil, the intestines of animals, and the like, and is mainlyused as a concept of genomes that explains identification of manymicroorganisms at one time using a sequencer to analyze non-culturedmicroorganisms. In particular, a metagenome does not refer to a genomeof one species, but refers to a mixture of genomes, including genomes ofall species of an environmental unit. This term originates from the viewthat, when defining one species in a process in which biology isadvanced into omics, various species as well as an existing speciesfunctionally interact with each other to form a complete species.Technically, it is the subject of techniques that analyze all DNAs andRNAs regardless of species using rapid sequencing to identify allspecies in one environment and verify interactions and metabolism.

In the present invention, the samples may be, but is not limited to,blood, urine, or stool.

According to another embodiment of the present invention, there isprovided a pharmaceutical composition for preventing or treating one ormore diseases selected from the group consisting of stomach cancer,colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, a brain tumor, chronic obstructive pulmonary disease,atopic dermatitis, irritable bowel syndrome, inflammatory enteritis,asthma, myocardial infarction, cardiomyopathy, variant angina, atrialfibrillation, a stroke, diabetes, Parkinson's disease, and depression,the pharmaceutical composition including vesicles derived from bacteriabelonging to the genus Proteus as an active ingredient.

According to another embodiment of the present invention, there isprovided an inhalant composition for preventing or treating one or morediseases selected from the group consisting of stomach cancer,colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, a brain tumor, chronic obstructive pulmonary disease,atopic dermatitis, irritable bowel syndrome, inflammatory enteritis,asthma, myocardial infarction, cardiomyopathy, variant angina, atrialfibrillation, a stroke, diabetes, Parkinson's disease, and depression,the inhalant composition including vesicles derived from bacteriabelonging to the genus Proteus as an active ingredient.

According to another embodiment of the present invention, there isprovided a composition for preventing or alleviating one or morediseases selected from the group consisting of stomach cancer,colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer,lung cancer, breast cancer, ovarian cancer, bladder cancer, prostatecancer, lymphoma, a brain tumor, chronic obstructive pulmonary disease,atopic dermatitis, irritable bowel syndrome, inflammatory enteritis,asthma, myocardial infarction, cardiomyopathy, variant angina, atrialfibrillation, a stroke, diabetes, Parkinson's disease, and depression,the composition including vesicles derived from bacteria belonging tothe genus Proteus as an active ingredient. The composition includes afood composition and a cosmetic composition.

The term “prevention” as used herein means all actions that inhibitcancer, a cardiovascular disease, a metabolic disease, aneuropsychiatric disease, an allergic-respiratory disease, and aninflammatory bowel disease or delay the onset thereof via administrationof the pharmaceutical composition according to the present invention.

The term “treatment” as used herein means all actions that alleviate orbeneficially change symptoms due to cancer, a cardiovascular disease, ametabolic disease, a neuropsychiatric disease, an allergic-respiratorydisease, and an inflammatory bowel disease via administration of thepharmaceutical composition according to the present invention.

The term “alleviation” as used herein means all actions that decrease atleast the degree of parameters related to conditions being treated,e.g., symptoms.

In one embodiment of the present invention, it was confirmed that, as aresult of evaluating in vivo absorption, distribution and excretionpatterns of bacteria and bacteria-derived vesicles after being orallyadministered to mice, the bacteria were not absorbed through theintestinal mucosa, while the vesicles were absorbed within 5 minutesafter administration and distributed systemically, and excreted throughthe kidneys, the liver, and the like (see Example 1).

In another embodiment of the present invention, bacterial metagenomicanalysis was performed using vesicles isolated from blood, urine, orstool samples of patients with stomach cancer, colorectal cancer, livercancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer, lymphoma, or a braintumor, and a normal control having age matched with that of thepatients. As a result, it was confirmed that vesicles derived frombacteria belonging to the genus Proteus was significantly reduced insamples of patients with stomach cancer, colorectal cancer, livercancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer,ovarian cancer, bladder cancer, prostate cancer, lymphoma, or a braintumor, as compared to that of samples of normal people (see Example 3).

In another embodiment of the present invention, bacterial metagenomicanalysis was performed using vesicles isolated from samples of patientswith diabetes, myocardial infarction, cardiomyopathy, atrialfibrillation, variant angina, or a stroke, and samples of normal peoplehaving age matched with that of the patients. As a result, it wasconfirmed that vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the samples of patients withdiabetes, myocardial infarction, cardiomyopathy, variant angina, atrialfibrillation, or a stroke, as compared to that of the samples of normalpeople (see Example 4).

In another embodiment of the present invention, bacterial metagenomicanalysis was performed using vesicles isolated from samples of patientswith Parkinson's disease or depression, and samples of normal peoplehaving age matched with that of the patients. As a result, it wasconfirmed that vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the samples of patients withParkinson's disease or depression, as compared to the samples of normalpeople (see Example 5).

In another embodiment of the present invention, bacterial metagenomicanalysis was performed using vesicles isolated from samples of patientswith atopic dermatitis, asthma, or chronic obstructive pulmonarydisease, and samples of normal people having age and gender matched withthose of the patients. As a result, it was confirmed that vesiclesderived from bacteria belonging to the genus Proteus was significantlyreduced in the samples of patients with atopic dermatitis, asthma, orchronic obstructive pulmonary disease, as compared to that of thesamples of normal people (see Example 6).

In another embodiment of the present invention, bacterial metagenomicanalysis was performed using vesicles isolated from samples of patientswith irritable bowel syndrome or inflammatory enteritis, and normalpeople having age matched with that of the patients. As a result, it wasconfirmed that vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the samples of patients withirritable bowel syndrome or inflammatory enteritis, as compared to thatof the samples of normal people (see Example 7).

In another embodiment of the present invention, as a result of furtherhaving conducted studies to analyze the characteristics of vesiclesderived from bacteria belonging to the genus Proteus, i.e., Proteusmirabilis, based on the results of the above-described examples, it wasconfirmed that the vesicles had an average diameter of less than 200 nm,preferably 41.40±5.03 nm, and were spherical (see Example 8).

In another embodiment of the present invention, as a result ofevaluating the secretion of an inflammatory mediator after Proteusmirabilis stains isolated from people were cultured, and thenmacrophages were treated with Proteus mirabilis-derived vesicles atvarious concentrations and treated with E. coli-derived vesicles, whichare causative factors of an inflammatory disease, a metabolic disease,and cancer, to examine whether vesicles secreted from culture broths ofthe Proteus mirabilis strains have an anti-inflammatory effect, it wasconfirmed that the secretion of TNF-α due to E. coli-derived vesicleswas efficiently inhibited in all the cases of Proteus mirabilis-derivedvesicles regardless of the origin of Proteus mirabilis (see Example 9).

In another embodiment of the present invention, to evaluate whether anactive material of Proteus mirabilis, which exhibits ananti-inflammatory effect, is a low-molecular-weight compound or proteinincluded in vesicles, Proteus mirabilis-derived vesicles wereheat-treated or acid-treated. Subsequently, as a result of evaluatingthe effect of heat-treated or acid-treated vesicles on the secretion ofTNF-α after being administered to macrophages prior to treatment with E.coli-derived vesicles, it was confirmed that the secretion of TNF-α dueto E. coli-derived vesicles was efficiently inhibited in both cases ofthe heat-treated or acid-treated Proteus mirabilis-derived vesicles (seeExample 10).

In another embodiment of the present invention, to evaluate whethervesicles secreted from culture broths of Proteus mirabilis strainsexhibit an anti-cancer therapeutic effect, cancer disease models wereprepared by subcutaneously injecting a cancer cell line, and as a resultof measuring the size of cancer tissue for 20 days after the Proteusmirabilis-derived vesicles were orally administered to mice from 4 daysbefore treatment, it was confirmed that the size of cancer tissue wassignificantly reduced in the case in which the vesicles wereadministered, as compared to that of a control (see Example 11).

The pharmaceutical composition according to the present inventionincludes vesicles derived from bacteria belonging to the genus Proteusas an active ingredient, and may include a pharmaceutically acceptablecarrier. The pharmaceutically acceptable carrier includes carrierscommonly used for formulation, e.g., saline, sterilized water, Ringer'ssolution, buffered saline, cyclodextrin, a dextrose solution, amaltodextrin solution, glycerol, ethanol, liposomes, and the like, butthe present invention is not limited thereto, and if needed, may furtherinclude other general additives such as antioxidants, buffer solutions,and the like. In addition, preparations for injection, such as aqueoussolutions, suspensions, emulsions, and the like, pills, capsules,granules, or tablets may be formulated by additionally adding a diluent,a dispersant, a surfactant, a binder, a lubricant, or the like. Withregards to suitable pharmaceutically acceptable carriers andformulations, preparations may be preferably formulated according toeach ingredient by using a method disclosed in the Remington'sreference. Preparations of the pharmaceutical composition of the presentinvention are not particularly limited, but the pharmaceuticalcomposition may be formulated into the form of injections, inhalants,external preparations for skin, oral ingestions, or the like.

The pharmaceutical composition of the present invention may beadministered orally or parenterally (e.g., intravenous administration,subcutaneous administration, intradermal administration, intranasaladministration, and intra-airway administration) according to a targetmethod, and a suitable dose thereof may vary depending on the conditionand body weight of a patient, the severity of disease, the type of drug,administration routes, and administration time, and may be appropriatelyselected by those of ordinary skill in the art.

The composition according to the present invention is administered in apharmaceutically effective amount. The term “pharmaceutically effectiveamount” as used herein refers to an amount sufficient to treat diseasesat a reasonable benefit/risk ratio applicable to medical treatment, andan effective dosage level may be determined according to factorsincluding the type of disease of a patient, the severity of disease,drug activity, sensitivity to a drug, administration time,administration routes, excretion rate, treatment period, andsimultaneously used drugs, and other factors well known in the medicalfield. The composition according to the present invention may beadministered as an individual therapeutic agent or in combination withother therapeutic agents, may be administered consecutively orsimultaneously with existing therapeutic agents, and may be administeredin a single dose or multiple doses. It is important to administer thecomposition in the minimum amount that enables achievement of themaximum effects without side effects in consideration of all theabove-described factors, and this may be easily determined by those ofordinary skill in the art.

In particular, an effective amount of the composition according to thepresent invention may vary according to the age, gender, and body weightof a patient. Generally, the composition may be administered in anamount of 0.001 mg to 150 mg, preferably, 0.01 mg to 100 mg, per bodyweight (1 kg) daily or every other day, or may be administered once orthree times a day. However, the dosage may be increased or decreasedaccording to administration routes, the severity of obesity, gender,body weight, age, and the like, and thus the dosage is not intended tolimit the scope of the present invention in any way.

In a food composition of the present invention, the active ingredientmay be added as is or used in combination with other foods or foodingredients, and it may be appropriately used according to a generalmethod. A mixing amount of the active ingredient may be appropriatelydetermined according to the purpose of use (for prevention oralleviation). Generally, when preparing foods or beverages, thecomposition of the present invention is added in an amount of 15 wt % orless, preferably 10 wt % or less, with respect to the amount of rawmaterials. However, in the case of long-term administration for healthand hygienic purposes or for health control, an amount less than theabove-described range may be used.

In addition to the active ingredient included as an essential ingredientin the food composition of the present invention at the indicated ratio,other ingredients of the food composition are not particularly limited,and the food composition may include various flavoring agents, naturalcarbohydrates, or the like as additional ingredients, like generalbeverages. Examples of the above-described natural carbohydrates includegeneral sugars, for example, monosaccharides such as glucose, fructose,and the like; disaccharides such as maltose, sucrose, and the like; andpolysaccharides such as dextrin, cyclodextrin, and the like, and sugaralcohols such as xylitol, sorbitol, erythritol, and the like. Examplesof sweeteners other than the above-described carbohydrates may includenatural sweeteners such as thaumatin and stevia extracts (e.g.,rebaudioside A, glycyrrhizin, and the like), and synthetic sweetenerssuch as saccharin, aspartame, and the like. The proportion of thenatural carbohydrate may be appropriately determined by those ofordinary skill in the art.

In addition, the food composition of the present invention may includevarious nutritional supplements, vitamins, minerals (electrolytes),flavors such as synthetic flavors, natural flavors, and the like,colorants and enhancers (cheese, chocolates, and the like), pectic acidand salts thereof, alginic acid and salts thereof, organic acids, aprotective colloid thickener, a pH adjuster, a stabilizer, apreservative, glycerin, alcohols, a carbonating agent used in carbonatedbeverages, and the like. These ingredients may be used alone or acombination thereof may be used. The proportion of these additives mayalso be appropriately selected by those of ordinary skill in the art.

A cosmetic composition of the present invention may include not onlyvesicles derived from bacteria belonging to the genus Proteus, but alsoingredients commonly used in cosmetic compositions, and may include, forexample, general adjuvants such as an antioxidant, a stabilizer, asolubilizing agent, vitamins, pigments, and dyes, and a carrier.

In addition, the composition of the present invention may furtherinclude, in addition to the vesicles derived from bacteria belonging tothe genus Proteus, an organic UV blocking agent that has long been usedwithin a range that does not adversely affect a skin protective effectby reaction with vesicles derived from bacteria belonging to the genusProteus. The organic UV blocking agent may be one or more selected fromthe group consisting of glyceryl PABA, drometrizole trisiloxane,drometrizole, digalloyl trioleate, disodium phenyl dibenzimidazoletetrasulfonate, diethyl hexyl butamidotriazone, diethyl amino hydroxylbenzoyl hexyl benzoate, DEA-methoxycinnamate, a mixture of lawsone anddihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol,4-methylbenzylidene camphor, menthyl anthranilate,benzophenone-3(oxybenzone), benzophenone-4,benzophenone-8(dioxybenzone), butylmethoxydibenzoylmethane,bisethylhexyloxyphenolmethoxyphenyltriazine, cinoxate,ethyldihydroxypropyl PABA, octocrylene, ethylhexyldimethyl PABA,ethylhexylmethoxycinnamate, ethylhexyl salicylate, ethylhexyl triazone,isoamyl-p-methoxycinnamate, polysilicon-15(dimethicodiethylbenzalmalonate), terephthalylidene dicamphor sulfonic acid and salts thereof,TEA-salicylate, and aminobenzoic acid (PABA).

Examples of products to which the cosmetic composition of the presentinvention may be added include cosmetics such as astringents, skinsofteners, nourishing toners, various creams, essences, packs,foundations, and the like, cleansing, face cleansers, soaps, treatments,beauty liquids, and the like. Particular preparations of the cosmeticcomposition of the present invention include a skin lotion, a skinsoftener, a skin toner, an astringent, a lotion, a milk lotion, amoisturizing lotion, a nourishing lotion, a massage cream, a nourishingcream, a moisturizing cream, a hand cream, an essence, a nourishingessence, a pack, a soap, a shampoo, a cleansing foam, a cleansinglotion, a cleansing cream, a body lotion, a body cleanser, an emulsion,a lipstick, a makeup base, a foundation, a press powder, a loose powder,an eye shadow, and the like.

According to an exemplary embodiment of the present invention, theamount of the vesicles derived from bacteria belonging to the genusProteus of the present invention ranges from 0.00001 wt % to 30 wt %,preferably 0.5 wt % to 20 wt %, and more preferably 1.0 wt % to 10 wt %,with respect to a total weight of the composition.

In the inhalant composition of the present invention, the activeingredient may be directly added to an inhalant or may be used incombination with other ingredients, and may be appropriately usedaccording to a general method. A mixing amount of the active ingredientmay be appropriately determined according to the purpose of use (forprevention or treatment).

Hereinafter, exemplary embodiments will be described to aid inunderstanding of the present invention. However, the following examplesare provided only to facilitate the understanding of the presentinvention and are not intended to limit the scope of the presentinvention.

MODE OF INVENTION Examples Example 1. Analysis of In Vivo Absorption,Distribution, and Excretion Patterns of Intestinal Bacteria andBacteria-Derived Vesicles

Experiments were conducted using the following method to evaluatewhether intestinal bacteria and bacteria-derived vesicles weresystemically absorbed through the gastrointestinal tract. Intestinalbacteria and intestinal bacteria-derived vesicles, which were labeledwith fluorescence, were administered to the gastrointestinal tract ofmice at a dose of 50 μg, and fluorescence was measured after 0 minute, 5minutes, 3 hours, 6 hours, and 12 hours.

As a result of observing the whole images of mice, as illustrated inFIG. 1A, while the bacteria were not systemically absorbed, thebacteria-derived vesicles were systemically absorbed at 5 minutes afteradministration, strong fluorescence was observed in the bladder at 3hours after administration, thereby confirming that the vesicles wereexcreted into the urinary tract. In addition, it was confirmed that thevesicles were present in the body until 12 hours (see FIG. 1A).

To evaluate the infiltration patterns of intestinal bacteria andintestinal bacteria-derived vesicles into various organs after beingsystemically absorbed, 50 μg of bacteria and bacteria-derived vesiclesthat were labeled with fluorescence were administered using theabove-described method, and then blood, the heart, the liver, thekidneys, the spleen, fat, and muscle were extracted 12 hours afteradministration.

As a result of observing fluorescence in the collected tissues, asillustrated in FIG. 1B, it was seen that the bacteria-derived vesicleswere distributed in blood, the heart, the lungs, the liver, the kidneys,the spleen, fat, and muscle, while the bacteria were not absorbed (seeFIG. 1B).

Example 2. Metagenomic Analysis of Bacteria-Derived Vesicles in ClinicalSamples

First, clinical samples such as blood, urine, stool, and the like werecentrifuged (3,500×g, 10 minutes, 4° C.) in a 10 ml tube to precipitatea floating material and only a supernatant was transferred into a new 10ml tube. Bacteria and impurities were removed therefrom using a 0.22 μmfilter, and then the resulting supernatant was transferred intocentripreigugal filters (50 kD), centrifuged at 1,500×g and 4° C. for 15minutes to remove a material having a size of less than 50 kD, andconcentrated up to 10 ml. Bacteria and impurities were removed againtherefrom using a 0.22 μm filter, and then subjected to ultrahigh speedcentrifugation at 150,000×g and 4° C. for 3 hours using a Type 90tirotor to remove a supernatant, and the agglomerated pellet was dissolvedwith phosphate buffered saline (PBS).

100 μl of the vesicles isolated using the above-described method wereboiled at 100° C. to release internal DNA out of the lipid, and thencooled on ice for 5 minutes. Then, to remove the remaining floatingmaterials, the resultant samples were centrifuged at 10,000×g and 4° C.for 30 minutes to collect only a supernatant. The amount of DNA was thenquantified using Nanodrop, and then PCR was performed on the extractedDNA using a pair of 16S rDNA primers shown in Table 1 below to confirmthe presence or absence of bacteria-derived DNA, thereby confirming thepresence of bacteria-derived genes in the extracted DNA.

TABLE 1 SEQ ID Primer Sequence NO. 16S rDNA 16S_V3_F5′-TCGTCGGCAGCGTCAGAT 1 GTGTATAAGAGACAGCCTACG GGNGGCWGCAG-3′ 16S_V4_R5′-GTCTCGTGGGCTCGGAGA 2 TGTGTATAAGAGACAGGACTA CHVGGGTATCTAATCC-3′

The DNA extracted using the above method was amplified using the pair of16S rDNA primers, and then sequenced (Illumina MiSeq sequencer), theresults were output in the form of a Standard Flowgram Format (SFF) fileand the SFF file was converted into a sequence file (.fasta) and anucleotide quality score file by using GS FLX software (v2.9), and thena reliability estimation for reads was identified and a portion with awindow (20 bps) average base call accuracy of less than 99% (Phred score<20) was removed. For operational taxonomy unit (OTU) analysis,clustering was performed according to sequence similarity using UCLUSTand USEARCH, the genus, family, order, class, and phylum were clusteredbased on the sequence similarities of 94%, 90%, 85%, 80%, and 75%,respectively, levels of the phylum, class, order, family, and genus ofrespective OTUs were classified, and bacteria having a sequencesimilarity of 97% or more at the genus level were profiled using aBLASTN and GreenGenes 16S RNA sequence database (108,453 sequences)(QIIME).

Example 3. Metagenomic Analysis of Bacteria-Derived Vesicles in ClinicalSamples of Patients with Cancer

Genes were extracted from vesicles present in stool samples of 55patients with stomach cancer and 99 normal people as a control by usingthe method of Example 2, metagenomic analysis was performed thereon, andthen the distribution of vesicles derived from bacteria belonging to thegenus Proteus was evaluated. As a result, it was confirmed that thevesicles derived from bacteria belonging to the genus Proteus wassignificantly reduced in the stool samples of the patients with stomachcancer, as compared to that of the stool samples of the normal people(normal people vs patients with stomach cancer: 1.1% vs 0.05%, p<0.001)(see FIG. 2 ).

In addition, genes were extracted from vesicles present in blood samplesof 67 patients with stomach cancer and 198 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theblood samples of the patients with stomach cancer, as compared to thatof the blood samples of the normal people (normal people vs patientswith stomach cancer: 1.3% vs 0.2%, p=<0.001) (see FIG. 2 ).

In addition, genes were extracted from vesicles present in urine samplesof 61 patients with stomach cancer and 120 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theurine samples of the patients with stomach cancer, as compared to thatof the urine samples of the normal people (normal people vs patientswith stomach cancer: 1.8% vs 0.1%, p<0.0001) (see FIG. 2 ).

In addition, genes were extracted from vesicles present in stool samplesof 38 patients with colorectal cancer and 55 normal people as a controlby using the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the stool samples of the patientswith colorectal cancer, as compared to that of the stool samples of thenormal people (normal people vs patients with colorectal cancer: 1.2% vs0.05%, p<0.001) (see FIG. 3 ).

In addition, genes were extracted from vesicles present in urine samplesof 38 patients with colorectal cancer and 38 normal people, who hadgender and age matched with those of the patients, as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theurine samples of the patients with colorectal cancer, as compared tothat of the urine samples of the normal people (normal people vspatients with colorectal cancer: 1.3% vs 0.06%, p<0.001) (see FIG. 3 ).

In addition, genes were extracted from vesicles present in blood samplesof 94 patients with liver cancer and 152 normal people as a control byusing the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the blood samples of the patientswith liver cancer, as compared to that of the blood samples of thenormal people (normal people vs patients with liver cancer: 0.7% vs0.01%, p<0.001) (see FIG. 4 ).

In addition, genes were extracted from vesicles present in blood samplesof 84 patients with bile duct cancer and 132 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theblood samples of the patients with bile duct cancer, as compared to thatof the blood samples of the normal people (normal people vs patientswith bile duct cancer: 0.7% vs 0.06%, p<0.001) (see FIG. 4 ).

In addition, genes were extracted from vesicles present in blood samplesof 191 patients with pancreatic cancer and 291 normal people as acontrol, metagenomic analysis was performed thereon, and then thedistribution of vesicles derived from bacteria belonging to the genusProteus was evaluated. As a result, it was confirmed that the vesiclesderived from bacteria belonging to the genus Proteus was significantlyreduced in the blood samples of the patients with pancreatic cancer, ascompared to that of the blood samples of the normal people (normalpeople vs patients with pancreatic cancer: 0.7% vs 0.06%, p<0.001) (seeFIG. 4 ).

In addition, genes were extracted from vesicles present in blood samplesof 318 patients with lung cancer and 234 normal people as a control byusing the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the blood samples of the patientswith lung cancer, as compared to that of the blood samples of the normalpeople (normal people vs patients with lung cancer: 0.7% vs 0.1%,p<0.001) (see FIG. 5 ).

In addition, genes were extracted from vesicles present in urine samplesof 127 patients with breast cancer and 220 normal people as a control byusing the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the urine samples of the patientswith breast cancer, as compared to that of the urine samples of thenormal people (normal people vs patients with breast cancer: 1.4% vs0.3%, p<0.001) (see FIG. 6 ).

In addition, genes were extracted from vesicles present in urine samplesof 136 patients with ovarian cancer and 136 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theurine samples of the patients with ovarian cancer, as compared to thatof the urine samples of the normal people (normal people vs patientswith ovarian cancer: 1.1% vs 0.1%, p<0.001) (see FIG. 6 ).

In addition, genes were extracted from vesicles present in blood samplesof 96 patients with bladder cancer and 184 normal people as a control byusing the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the blood samples of the patientswith bladder cancer, as compared to that of the blood samples of thenormal people (normal people vs patients with bladder cancer: 0.8% vs0.1%, p<0.001) (see FIG. 7 ).

In addition, genes were extracted from vesicles present in urine samplesof 95 patients with bladder cancer and 157 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theurine samples of the patients with bladder cancer, as compared to thatof the urine samples of the normal people (normal people vs patientswith bladder cancer: 1.9% vs 0.1%, p<0.000001) (see FIG. 7 ).

In addition, genes were extracted from vesicles present in urine samplesof 53 patients with prostate cancer and 159 normal people as a controlby using the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the urine samples of the patientswith prostate cancer, as compared to that of the urine samples of thenormal people (normal people vs patients with prostate cancer: 1.1% vs0.09%, p<0.000001) (see FIG. 8 ).

In addition, genes were extracted from vesicles present in blood samplesof 93 patients with lymphoma and 109 normal people as a control by usingthe method of Example 2, metagenomic analysis was performed thereon, andthen the distribution of vesicles derived from bacteria belonging to thegenus Proteus was evaluated. As a result, it was confirmed that thevesicles derived from bacteria belonging to the genus Proteus wassignificantly reduced in the blood samples of the patients withlymphoma, as compared to that of the blood samples of the normal people(normal people vs patients with lymphoma: 0.09% vs 0.00%, p<0.001) (seeFIG. 9 ).

In addition, genes were extracted from vesicles present in blood samplesof 84 patients with a brain tumor and 92 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theblood samples of the patients with a brain tumor, as compared to that ofthe blood samples of the normal people (normal people vs patients with abrain tumor: 0.09% vs 0.01%, p<0.01) (see FIG. 9 ).

Example 4. Metagenomic Analysis of Bacteria-Derived Vesicles in ClinicalSamples of Patients with Diabetes and Cardiovascular Disease

Genes were extracted from vesicles present in blood samples of 73patients with diabetes and 146 normal people as a control by using themethod of Example 2, metagenomic analysis was performed thereon, andthen the distribution of vesicles derived from bacteria belonging to thegenus Proteus was evaluated. As a result, it was confirmed that thevesicles derived from bacteria belonging to the genus Proteus wassignificantly reduced in the blood samples of the patients withdiabetes, as compared to that of the blood samples of the normal people(normal people vs patients with diabetes: 0.4% vs 0.01%, p<0.01) (seeFIG. 10 ).

In addition, genes were extracted from vesicles present in blood samplesof 57 patients with myocardial infarction and 163 normal people as acontrol by using the method of Example 2, metagenomic analysis wasperformed thereon, and then the distribution of vesicles derived frombacteria belonging to the genus Proteus was evaluated. As a result, itwas confirmed that the vesicles derived from bacteria belonging to thegenus Proteus was significantly reduced in the blood samples of thepatients with myocardial infarction, as compared to that of the bloodsamples of the normal people (normal people vs patients with myocardialinfarction: 0.4% vs 0.07%, p<0.01) (see FIG. 11 ).

In addition, genes were extracted from vesicles present in blood samplesof 72 patients with cardiomyopathy and 163 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theblood samples of the patients with cardiomyopathy, as compared to thatof the blood samples of the normal people (normal people vs patientswith cardiomyopathy: 0.4% vs 0.08%, p<0.01) (see FIG. 11 ).

In addition, genes were extracted from vesicles present in blood samplesof 69 patients with atrial fibrillation and 103 normal people as acontrol, metagenomic analysis was performed thereon, and then thedistribution of vesicles derived from bacteria belonging to the genusProteus was evaluated. As a result, it was confirmed that the vesiclesderived from bacteria belonging to the genus Proteus was significantlyreduced in the blood samples of the patients with atrial fibrillation,as compared to that of the blood samples of the normal people (normalpeople vs patients with atrial fibrillation: 0.1% vs 0.01%, p<0.01) (seeFIG. 11 ).

In addition, genes were extracted from vesicles present in blood samplesof 32 patients with variant angina and 32 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theblood samples of the patients with variant angina, as compared to thatof the blood samples of the normal people (normal people vs patientswith variant angina: 0.6% vs 0.1%, p<0.05) (see FIG. 11 ).

In addition, genes were extracted from vesicles present in blood samplesof 87 patients with a stroke and 92 normal people as a control,metagenomic analysis was performed thereon, and then the distribution ofvesicles derived from bacteria belonging to the genus Proteus wasevaluated. As a result, it was confirmed that the vesicles derived frombacteria belonging to the genus Proteus was significantly reduced in theblood samples of the patients with a stroke, as compared to that of theblood samples of the normal people (normal people vs patients with astroke: 0.3% vs 0.00%, p<0.05) (see FIG. 12 ).

Example 5. Metagenomic Analysis of Bacteria-Derived Vesicles in ClinicalSamples of Patients with Neuropsychiatric Disease

Genes were extracted from vesicles present in urine samples of 39patients with Parkinson's disease and 79 normal people as a control byusing the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the urine samples of the patientswith Parkinson's disease, as compared to that of the urine samples ofthe normal people (normal people vs patients with Parkinson's disease:0.6% vs 0.02%, p<0.0001) (see FIG. 13 ).

In addition, genes were extracted from vesicles present in urine samplesof 20 patients with depression and 20 normal people as a control byusing the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the urine samples of the patientswith depression, as compared to that of the urine samples of the normalpeople (normal people vs patients with depression: 1.7% vs 0.06%,p<0.01) (see FIG. 13 ).

Example 6. Metagenomic Analysis of Bacteria-Derived Vesicles in ClinicalSamples of Patients with Allergic or Respiratory Disease

Genes were extracted from vesicles present in blood samples of 27patients with atopic dermatitis and 138 normal people as a control byusing the method of Example 2, metagenomic analysis was performedthereon, and then the distribution of vesicles derived from bacteriabelonging to the genus Proteus was evaluated. As a result, it wasconfirmed that the vesicles derived from bacteria belonging to the genusProteus was significantly reduced in the blood samples of the patientswith atopic dermatitis, as compared to that of the blood samples of thenormal people (normal people vs patients with atopic dermatitis: 2.0% vs0.06%, p<0.00001) (see FIG. 14 ).

In addition, genes were extracted from vesicles present in blood samplesof 291 patients with asthma, 207 patients with chronic obstructivepulmonary disease (COPD), and 291 normal people as a control by usingthe method of Example 2, metagenomic analysis was performed thereon, andthen the distribution of vesicles derived from bacteria belonging to thegenus Proteus was evaluated. As a result, it was confirmed that thevesicles derived from bacteria belonging to the genus Proteus wassignificantly reduced in the blood samples of the patients with asthmaor chronic obstructive pulmonary disease, as compared to that of theblood samples of the normal people (normal people vs patients withasthma: 0.7% vs 0.08%, p<0.01; normal people vs patients with chronicobstructive pulmonary disease: 0.7% vs 0.07%, p<0.01) (see FIG. 15 ).

Example 7. Metagenomic Analysis of Bacteria-Derived Vesicles in ClinicalSamples of Patients with Inflammatory Bowel Disease

Genes were extracted from vesicles present in stool samples of 57patients with irritable bowel syndrome (IBS) and 58 normal people as acontrol by using the method of Example 2, metagenomic analysis wasperformed thereon, and then the distribution of vesicles derived frombacteria belonging to the genus Proteus was evaluated. As a result, itwas confirmed that the vesicles derived from bacteria belonging to thegenus Proteus was significantly reduced in the stool samples of thepatients with irritable bowel syndrome, as compared to that of the stoolsamples of the normal people (normal people vs patients with irritablebowel syndrome: 1.9% vs 0.00%, p<0.00001) (see FIG. 16 ).

In addition, genes were extracted from vesicles present in stool samplesof 91 patients with inflammatory enteritis and 99 normal people as acontrol by using the method of Example 2, metagenomic analysis wasperformed thereon, and then the distribution of vesicles derived frombacteria belonging to the genus Proteus was evaluated. As a result, itwas confirmed that the vesicles derived from bacteria belonging to thegenus Proteus was significantly reduced in the stool samples of thepatients with inflammatory enteritis, as compared to that of the stoolsamples of the normal people (normal people vs patients withinflammatory enteritis: 1.9% vs 0.00%, p<0.00001) (see FIG. 16 ).

Example 8. Isolation of Vesicles from Proteus mirabilis Culture Brothand Analysis of Characteristics thereof

Based on the results of Examples 2 to 7, to evaluate the function ofvesicles derived from bacteria belonging to the genus Proteus, theProteus mirabilis strain, which is one species of bacteria belonging tothe genus Proteus, was cultured and then vesicles were isolatedtherefrom, and characteristics of the vesicles were analyzed. TheProteus mirabilis strain isolated from clinical samples of people wascultured in a brain heart infusion (BHI) medium in an anaerobic chamberat 37° C. until absorbance (OD₆₀₀) reached 1.0 to 1.5, and thensub-cultured. Subsequently, a Proteus mirabilis strain-free mediumsupernatant was collected and centrifuged at 10,000×g and 4° C. for 15minutes, and the resulting supernatant was filtered using a 0.45 μmfilter, and then concentrated to a volume of 200 ml through a 100 kDahollow filter membrane by using a QuixStand benchtop system (GEHealthcare, UK). Thereafter, the concentrated supernatant was filteredagain with a 0.22 μm filter. Proteins were then quantified using BCAassay, and the following experiments were performed on the obtainedvesicles, and vesicles were isolated from a culture broth of Proteusmirabilis cultured according to the above method, and then the shape andsize thereof were evaluated using an electron microscope.

As a result, as illustrated in FIG. 17A, it was confirmed that thevesicles isolated from the Proteus mirabilis culture broth werespherical and had a size of less than 200 nm, and it was confirmedthrough dynamic light scattering measurement results shown in FIG. 17Bthat the vesicles had a size of 37.8±13.5 nm.

Example 9. Anti-Inflammatory Effect of Proteus mirabilis-DerivedVesicles

To examine an effect of Proteus mirabilis-derived vesicles on thesecretion of inflammatory mediators in inflammatory cells, Raw 264.7cells, which is a mouse macrophage line, were treated with Proteusmirabilis-derived vesicles (P. mirabilis EV) at various concentrations(0.1, 1, or 10 μg/ml), and then treated with E. coli-derived vesicles(E. coli EV), which are pathogenic vesicles, and the secretion amountsof inflammatory mediators (IL-6, TNF-α, and the like) were measured.

More specifically, Raw 264.7 cells were dispensed into a 24-well cellculture plate at a density of 1×10⁵ cells/well, and then cultured in aDulbecco's Modified Eagle's Media (DMEM) complete medium for 24 hours.Subsequently, the culture supernatant was collected in a 1.5 ml tube andcentrifuged at 3,000×g for 5 minutes, and the supernatant was collectedand stored at 4° C., followed by ELISA analysis.

For ELISA analysis, the capture antibody was diluted with phosphatebuffered saline (PBS) and the diluted solution was dispensed in 50 μlaliquots into a 96-well polystyrene plate in accordance with a workingconcentration, and then allowed to react at 4° C. overnight.Subsequently, the sample was washed twice with 100 μl of a PBST (0.05%Tween-20-containing PBS) solution, and then an RD (1% bovine serumalbumin (BSA)-containing PBST) solution was dispensed in 100 μl aliquotsinto the plate, followed by blocking at room temperature for 1 hour andwashing twice again with 100 μl of PBST, and then the sample and astandard were dispensed in 50 μl aliquots in accordance withconcentration and allowed to react at room temperature for 2 hours. Thesample and the standard were washed twice again with 100 μl of PBST, andthen the detection antibody was diluted with RD, and the dilutedsolution was dispensed into 50 μl aliquots in accordance with a workingconcentration and allowed to react at room temperature for 2 hours. Thesample and the standard were washed twice again with 100 μl of PBST, andthen streptavidin-horseradish peroxidase (HRP) was diluted in RD to1/200, and the diluted solution was dispensed in 50 μl aliquots andallowed to react at room temperature for 30 minutes. Lastly, the sampleand the standard were washed three times with 100 μl of PBST, and then asolution prepared by mixing a 3,3,5,5-tetramethylbenzidine (TMB)substrate and 0.04% oxygenated water in a ratio of 1:1 was dispensed in50 μl aliquots. Thereafter, color developing was waited for and whencolor was developed after 5 minutes to 20 minutes, a 1M sulfuric acidsolution was dispensed in 50 μl aliquots, the reaction was stopped, andabsorbance at 450 nm was measured using a Synergy™ HT multi-detectionmicroplate reader (BioTek, USA).

As a result, it was confirmed that the secretion of IL-6 and TNF-α dueto E. coli-derived vesicles was significantly inhibited in the case ofpretreatment with Proteus mirabilis-derived vesicles (see FIGS. 18A and18B).

In addition, to evaluate effects of isoforms of Proteus mirabilis on theanti-inflammatory effect of Proteus mirabilis-derived vesicles,pretreatment with vesicles derived from 5 Proteus mirabilis strains(PMR201, PMR202, PMR203, PMR204, and PMR205) isolated from people, atvarious concentrations was performed for 12 hours, followed by treatmentwith 1 μg/ml of E. coli-derived vesicles, which are pathogenic vesicles,for 12 hours. The culture supernatant was collected in a 1.5 ml tube andcentrifuged at 3,000×g for 5 minutes, and the supernatant was collectedand stored at 4° C., followed by ELISA analysis.

As a result, it was confirmed that similarly to the case of Proteusmirabilis reference strain (PMR101)-derived vesicles, the secretion ofTNF-α due to E. coli-derived vesicles was significantly inhibited in thecase of pretreatment with the vesicles derived from isolated Proteusmirabilis strains (see FIG. 19 ). In particular, it was confirmed thatthe inhibitory effect on the secretion of TNF-α was greater in the caseof pretreatment with Proteus mirabilis-derived vesicles than in the caseof vesicles derived from Lactobacillus plantarum, which is an effectivemicroorganism control (see FIG. 19 ).

This indicates that vesicles derived from isolated Proteus mirabilisstrains were able to efficiently inhibit inflammatory responses inducedby pathogenic vesicles such as E. coli-derived vesicles, regardless ofisoforms thereof.

Example 10. Effect of Heat-Treated or Acid-Treated Proteusmirabilis-Derived Vesicles on Anti-Inflammatory Activity

Through Example 9, the anti-inflammatory effects of vesicles derivedfrom the Proteus mirabilis reference strain and isolated Proteusmirabilis strains were confirmed, and further, the stability of thevesicles and properties of the active ingredient were specificallyinvestigated. For this, macrophages (Raw 264.7) were pretreated withthree types of Proteus mirabilis-derived vesicles (PMR101, PMR202, andPMR205) that had been boiled at 100° C. for 10 minutes or subjected toacid treatment (pH 2.0) for 10 minutes to evaluate anti-inflammatoryeffects thereof. As a result, it was confirmed that theanti-inflammatory effects of the Proteus mirabilis-derived vesicles weremaintained even after boiling at 100° C. or acid treatment (see FIG. 20). This indicates that Proteus mirabilis-derived vesicles are stable ata high temperature and a high acid level, and this also indicates that acomponent of Proteus mirabilis-derived vesicles which exhibits ananti-inflammatory effect is not a protein.

Example 11. Anticancer Effect of Proteus mirabilis-Derived Vesicles

Through the above examples, vesicles derived from bacteria belonging tothe genus Proteus was significantly reduced in clinical samples ofpatients with cancer, as compared to that of normal people, and further,an anticancer effect of the vesicles was specifically investigated. Forthis, as depicted in FIG. 21A, cancer models were prepared by orallyadministering Proteus mirabilis-derived vesicles to male 6-week-oldC57BL/6 mice and subcutaneously injecting a cancer cell line (CT26cells) into the mice on day 4 after administration. Until day 20 afterthe cancer cell line was administered, the size of cancer tissue wasmeasured to evaluate cancer therapeutic effects. As a result, the sizeof cancer tissue was significantly reduced in the mice orallyadministered the vesicles, as compared to that of a group orallyadministered normal saline (see FIG. 21B).

The foregoing description of the present invention is provided forillustrative purposes only, and it will be understood by those ofordinary skill in the art to which the present invention pertains thatthe present invention may be easily modified into other particular formswithout changing the technical spirit or essential characteristics ofthe present invention. Thus, the above-described embodiments should beconstrued as being provided for illustrative purposes only and not forpurposes of limitation.

INDUSTRIAL APPLICABILITY

Vesicles derived from bacteria belonging to the genus Proteus, accordingto the present invention, can be used in a method of diagnosing variousdiseases, such as cancers such as stomach cancer, colorectal cancer,liver cancer, bile duct cancer, pancreatic cancer, lung cancer, breastcancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, abrain tumor, and the like; cardiovascular diseases such as myocardialinfarction, cardiomyopathy, atrial fibrillation, variant angina, astroke, and the like; metabolic diseases such as diabetes, and the like;neuropsychiatric diseases such as Parkinson's disease, depression, andthe like; allergic-respiratory diseases such as atopic dermatitis,asthma, chronic obstructive pulmonary disease, and the like; orinflammatory bowel diseases such as irritable bowel syndrome,inflammatory enteritis, and the like, and thus it is economical and itis anticipated that the vesicles can be applied to various applicationssuch as compositions for prevention, treatment, and/or alleviation,e.g., foods, inhalants, cosmetic, drugs, and the like. Therefore, thevesicles can be usefully used in various industrial fields such asmedical, functional food, and cosmetic industries.

The invention claimed is:
 1. A method of reducing inflammation mediatedby TNF-α or IL-6, comprising administering a composition comprising aneffective amount of vesicles isolated from bacteria belonging to Proteusmirabilis to the subject having the inflammation.
 2. The method of claim1, wherein the composition is a pharmaceutical composition, a cosmeticcomposition, or a food composition.
 3. The method of claim 1, whereinthe vesicles have an average diameter of 10 to 200 nm.
 4. The method ofclaim 1, wherein the vesicles are naturally secreted or artificiallyisolated from the bacteria.
 5. The method of claim 1, wherein thevesicles are naturally secreted from Proteus mirabilis.
 6. The method ofclaim 1, wherein the composition is an inhalant composition.